Study on the inhibitory effect of antisense phosplhorothioate oligodeoxynucleotide on coxsackie virus B3 replication in vitro

Abstract

Objective To investigate the antiviral effect of antisense phosphorothioate oligodeoxynucleotide (AODN) on CVB3 repli-cation in vitro. Methods In this study, 21met antisense phosphorothioate oligodeoxynucleotide which complemented to nt 581 - 601 in 5'NCR of Coxssckie Virus B3 (CVB3) RNA was used to investigate antiviral activity in Vero cells as a specific inhibitor of CVB3 replication. Spe-cific AODN of CVB3 RNA, sense oligodeoxynucleotide (SODN) and randomized nonsense - sequence RODN were synthesized for effect com-parison among them. Vero cells infected by CVB3 were transfected with different concentrations of AODN mediated by Lipofectamlne regeant,so that the cells could take in more AODN. Cell control and viral control were set up. The inhibitory effect of AODN on CVB3 replication wasevaluated with a number of variables, including inhibitory rates of cytopathic effect (CPE), cell survival rates by MTT assay, inhibitory ratesof CVB3 antigen by ELlSA, inhibitory rates of RNA by dot blotting and 50 % tissue culture infective dose (TCID5). Results The specificAODN could significantiy inhibit CPE of CVB3 - infected Vero cells, decrease the production of antigen and RNA of CVB3 and viral titres, andincrease cell survival rates, in a dose - dependent manner. The strongest inhibitory effect appeared at 48 hours after transfection, the most ef-fective concentration of AODN was 10 μmol/L. On the other hand, 10 μmol/L SODN also showed weaker inhibitory effect on CPE of CVB3 -infected cells, but no antiviral effect of 10 μmol/L RODN was shown. AODN showed no- inhibition on HSV - 1 replication, but inhibitedsome entroviruses such as CVB5, Polio- 1 and Echo- 6 to some extent. Conclusions These results indicate that nt 581 -601 in 5'NCR ofCVB3 RNA may play an important role in regulating CV B3 replication and AODN may have inhibitory effect on CVB3 replication. Key words: Antisense oligodeoxynuclcetlde; Coxsackie virus B3; Vero cells